The endothelium is an integral mediator of vascular homeostasis and a dysfunctional endothelium is now recognized as an early marker of atherosclerosis. Importantly, the non-invasive measurement of endothelial function by flow-mediated dilation (FMD) predicts future cardiovascular events. However, the appropriate method of its assessment and the mechanisms that govern FMD are still poorly understood. We investigated alternative parameters and methods of FMD measurement in healthy volunteers and cardiovascular disease patients. We found time to peak FMD to be highly variable both within and between individuals. Accordingly, continuous arterial diameter measurement post-cuff release was more sensitive in discriminating between health and disease compared to the measurement of diameter at 60’’ post-cuff release. Reperfusion to an ischemic tissue can paradoxically contribute to endothelial dysfunction development and further tissue damage, in a phenomenon known as ischemia and reperfusion (IR) injury. Previous exposure to sublethal ischemia (ischemic preconditioning (IPC)) can reduce sensitivity to IR injury and pharmacologic agents have since been shown to mimic this response. Using the FMD technique, we investigated various preconditioning strategies to prevent IR-induced endothelial dysfunction in the forearm vasculature of healthy volunteers. The sodium-hydrogen exchanger inhibitor amiloride and the angiotensin-converting enzyme inhibitor captopril were found not to provide endothelial protection from IR. In contrast, potent protection from IR-induced endothelial dysfunction was observed during the high-estrogen, late follicular phase of the menstrual cycle in pre-menopausal women. Finally, daily episodes of IPC were found to provide endothelial protection equipotent to an acute episode of IPC. The findings from the FMD methodological study highlight the importance of continuous arterial diameter measurement post-cuff deflation, and provide mechanistic insight that may contribute to measurement standardization and normalization. The results of the preconditioning studies improve our understanding of potential approaches to mitigate the detrimental effects of IR on the endothelium in humans.

Myocardial ischemia-reperfusion injury occurs following the majority of cardiac events including myocardial stenosis and heart surgeries. As reactive oxygen species are one of the major contributors to ischemia-reperfusion injury, strategies to prevent their effects may be directed towards enhancing the antioxidant capacity of cells. Polyphenols, and in a more specific category, flavonoids are strong antioxidants, while possessing other biological activities such as anti-apoptotic, anti-inflammatory, and vasodilatory effects. <p>I hypothesized that flavonoids are able to reduce ischemia-reperfusion-induced cell death through multiple mechanisms including reduction of oxidative stress and induction of cellular antioxidant enzymes. The hypothesis was tested in<i> in vitro</i> and <i> in vivo</i> phases.<p>In the first phase of the studies, rat embryonic ventricular H9c2 cells were treated with various concentrations of polyphenols with or without ascorbate for 1-3 days before induction of ischemia and reperfusion. Ischemia was induced by exposure of the cells to a non-glucose containing solution bubbled with nitrogen, and reperfusion by returning the regular medium containing the corresponding polyphenols and/or ascorbate. Cell viability measurements using the MTT assay or counting acridine orange-stained cells showed that the best protection against cell death was given by catechin (44-58 %), epigallocatechin gallate (48%), proanthocyanidins (44%), and ascorbic acid (57-92%). A low concentration (10 µM) of catechin was more effective with a long-term (2 days) incubation time (64%), while a higher concentration (50 µM) could exert benefit even after 1 h pre-treatment (98%). Quercetin, resveratrol, cyanidin, and delphinidin displayed almost no protection. <P>In the second part of the in vitro study, H9c2 cells were treated with 350 to 450 µM tert-butyl hydroperoxide for 24 h after pre-incubation with various concentrations of polyphenols with or without ascorbate for either short (1 h) or prolonged (3 days) periods. Unlike in the ischemia-reperfusion experiments, 3 days pre-treatment with polyphenols did not protect and often caused cytotoxicity. In short-term (1 h) pre-treatments, the best protection was obtained with 50 µM quercetin (95%), 50 µM epigallocatechin gallate (66%), and 100 µM catechin (28%). Pre-treatment with ascorbic acid (100 µM) with or without polyphenols did not improve cell survival except in one case where it enhanced cytoprotection by epigallocatechin gallate.<p>The second phase of the studies was performed with isolated rat hearts. Rats were fed diets containing broccoli sprouts (2%), saskatoon berries (5%), or green tea extract (0.25%) for 10 days before induction of global ischemia for 20 min and reperfusion for 2 h. Broccoli sprouts decreased cell death in ischemic-reperfused hearts as assessed by caspase-3 activity (86%) and DNA fragmentation (78 %), attenuated oxidative damage as detected by lower thiobarbituric acid reactive substances (TBARS) (116%) and preserved aconitase activity (82%). Green tea extract prevented apoptosis in hearts as detected by caspase-3 activity (85%), but did not inhibit DNA fragmentation. Berries showed lower TBARS (73%). None of the feedings significantly prevented necrosis as evaluated by the release of lactate dehydrogenase into the coronary effluents, improved coronary flow, or increased heart glutathione.<p>Green tea extract was the only intervention capable of preserving the activity of glutamate cysteine ligase (78%) and quinone reductase (147%) in hearts. The sprouts group was the only group which induced these same enzymes in liver (40 and 44 %, respectively), as it was the only intervention which elevated total liver glutathione (12%). None of the interventions changed heme oxygenase-1 protein levels. Assessment of total polyphenol content revealed that broccoli sprouts had the lowest and green tea extract had the highest amount of polyphenols among the three plant materials, suggesting that the protection exhibited by broccoli sprouts was unlikely to be due to the polyphenols. <p>In conclusion, flavonoids and flavonoid-rich foods can strengthen the cellular ability to fight against oxidative stress. A part of this effect could be due to their direct antioxidant activity, while in prolonged applications they may also activate cellular pathways to promote endogenous antioxidant defences of cells. Application of low doses of flavonoids and consumption of flavonoid-rich plants in long-term ensures their effectiveness while avoiding possible toxicity. However, plants such as broccoli sprouts may have other chemical ingredients bearing biological properties which may help cells to survive states of oxidative stress.

The endothelium is an integral mediator of vascular homeostasis and a dysfunctional endothelium is now recognized as an early marker of atherosclerosis. Importantly, the non-invasive measurement of endothelial function by flow-mediated dilation (FMD) predicts future cardiovascular events. However, the appropriate method of its assessment and the mechanisms that govern FMD are still poorly understood. We investigated alternative parameters and methods of FMD measurement in healthy volunteers and cardiovascular disease patients. We found time to peak FMD to be highly variable both within and between individuals. Accordingly, continuous arterial diameter measurement post-cuff release was more sensitive in discriminating between health and disease compared to the measurement of diameter at 60’’ post-cuff release. Reperfusion to an ischemic tissue can paradoxically contribute to endothelial dysfunction development and further tissue damage, in a phenomenon known as ischemia and reperfusion (IR) injury. Previous exposure to sublethal ischemia (ischemic preconditioning (IPC)) can reduce sensitivity to IR injury and pharmacologic agents have since been shown to mimic this response. Using the FMD technique, we investigated various preconditioning strategies to prevent IR-induced endothelial dysfunction in the forearm vasculature of healthy volunteers. The sodium-hydrogen exchanger inhibitor amiloride and the angiotensin-converting enzyme inhibitor captopril were found not to provide endothelial protection from IR. In contrast, potent protection from IR-induced endothelial dysfunction was observed during the high-estrogen, late follicular phase of the menstrual cycle in pre-menopausal women. Finally, daily episodes of IPC were found to provide endothelial protection equipotent to an acute episode of IPC. The findings from the FMD methodological study highlight the importance of continuous arterial diameter measurement post-cuff deflation, and provide mechanistic insight that may contribute to measurement standardization and normalization. The results of the preconditioning studies improve our understanding of potential approaches to mitigate the detrimental effects of IR on the endothelium in humans.

Myocardial ischemia-reperfusion injury occurs following the majority of cardiac events including myocardial stenosis and heart surgeries. As reactive oxygen species are one of the major contributors to ischemia-reperfusion injury, strategies to prevent their effects may be directed towards enhancing the antioxidant capacity of cells. Polyphenols, and in a more specific category, flavonoids are strong antioxidants, while possessing other biological activities such as anti-apoptotic, anti-inflammatory, and vasodilatory effects. <p>I hypothesized that flavonoids are able to reduce ischemia-reperfusion-induced cell death through multiple mechanisms including reduction of oxidative stress and induction of cellular antioxidant enzymes. The hypothesis was tested in<i> in vitro</i> and <i> in vivo</i> phases.<p>In the first phase of the studies, rat embryonic ventricular H9c2 cells were treated with various concentrations of polyphenols with or without ascorbate for 1-3 days before induction of ischemia and reperfusion. Ischemia was induced by exposure of the cells to a non-glucose containing solution bubbled with nitrogen, and reperfusion by returning the regular medium containing the corresponding polyphenols and/or ascorbate. Cell viability measurements using the MTT assay or counting acridine orange-stained cells showed that the best protection against cell death was given by catechin (44-58 %), epigallocatechin gallate (48%), proanthocyanidins (44%), and ascorbic acid (57-92%). A low concentration (10 µM) of catechin was more effective with a long-term (2 days) incubation time (64%), while a higher concentration (50 µM) could exert benefit even after 1 h pre-treatment (98%). Quercetin, resveratrol, cyanidin, and delphinidin displayed almost no protection. <P>In the second part of the in vitro study, H9c2 cells were treated with 350 to 450 µM tert-butyl hydroperoxide for 24 h after pre-incubation with various concentrations of polyphenols with or without ascorbate for either short (1 h) or prolonged (3 days) periods. Unlike in the ischemia-reperfusion experiments, 3 days pre-treatment with polyphenols did not protect and often caused cytotoxicity. In short-term (1 h) pre-treatments, the best protection was obtained with 50 µM quercetin (95%), 50 µM epigallocatechin gallate (66%), and 100 µM catechin (28%). Pre-treatment with ascorbic acid (100 µM) with or without polyphenols did not improve cell survival except in one case where it enhanced cytoprotection by epigallocatechin gallate.<p>The second phase of the studies was performed with isolated rat hearts. Rats were fed diets containing broccoli sprouts (2%), saskatoon berries (5%), or green tea extract (0.25%) for 10 days before induction of global ischemia for 20 min and reperfusion for 2 h. Broccoli sprouts decreased cell death in ischemic-reperfused hearts as assessed by caspase-3 activity (86%) and DNA fragmentation (78 %), attenuated oxidative damage as detected by lower thiobarbituric acid reactive substances (TBARS) (116%) and preserved aconitase activity (82%). Green tea extract prevented apoptosis in hearts as detected by caspase-3 activity (85%), but did not inhibit DNA fragmentation. Berries showed lower TBARS (73%). None of the feedings significantly prevented necrosis as evaluated by the release of lactate dehydrogenase into the coronary effluents, improved coronary flow, or increased heart glutathione.<p>Green tea extract was the only intervention capable of preserving the activity of glutamate cysteine ligase (78%) and quinone reductase (147%) in hearts. The sprouts group was the only group which induced these same enzymes in liver (40 and 44 %, respectively), as it was the only intervention which elevated total liver glutathione (12%). None of the interventions changed heme oxygenase-1 protein levels. Assessment of total polyphenol content revealed that broccoli sprouts had the lowest and green tea extract had the highest amount of polyphenols among the three plant materials, suggesting that the protection exhibited by broccoli sprouts was unlikely to be due to the polyphenols. <p>In conclusion, flavonoids and flavonoid-rich foods can strengthen the cellular ability to fight against oxidative stress. A part of this effect could be due to their direct antioxidant activity, while in prolonged applications they may also activate cellular pathways to promote endogenous antioxidant defences of cells. Application of low doses of flavonoids and consumption of flavonoid-rich plants in long-term ensures their effectiveness while avoiding possible toxicity. However, plants such as broccoli sprouts may have other chemical ingredients bearing biological properties which may help cells to survive states of oxidative stress.

Objective: Ischemic spinal cord injury is a serious complication of aortic surgery. The mechanism underlying ischemic preconditioning (IPC) protection against spinal cord ischemia/reperfusion (I/R) injury is unclear. We investigated the role of spinal cord autoregulation in tolerance to spinal cord I/R injury induced by IPC. Although the extracellular signal-regulated kinases 1 and 2 (ERK1/2) are generally regarded as related to cell survival and proliferation, increasing evidence suggests that the role of the ERK1/2 pathway in I/R injury is contributory to inflammation. We investigated the effect of blocking ERK1/2 pathway to inhibit inflammation reaction in tolerance to spinal cord I/R injury. Methods: In the part 1 study, Sprague-Dawley rats were randomly assigned to 4 groups. IPC (P) group animals received IPC by temporary thoracic aortic occlusion (AO) with a 2-F Fogarty arterial embolectomy catheter for 3 min. I/R injury (I/R) group animals were treated with blood withdrawal and temporary AO for 12 min, and shed blood reinfusion at the end of the procedures. (P+I/R) group animals received IPC, followed by 5 min reperfusion, and then I/R procedures for 12 min. Sham (S) group animals received anesthesia and underwent surgical preparation only. Neurological functions were evaluated, and lumbar segments were harvested for histopathological examination. To evaluate the role of autoregulation in IPC, spinal cord blood flow and tissue oxygenation were continuously monitored throughout the procedure duration. In the part 2 study, spinal cord ischemia rats was induced by occluding the thoracic descending aorta with a balloon catheter introduced through a femoral artery, accompanied by concomitant exsanguinations. Rats in the control group were given dimethyl sulfoxide (vehicle) before undergoing spinal cord ischemia/reperfusion injury. In the U0126-treated group, rats were pretreated with an inhibitor of ERK1/2, U0126, to inhibit ERK1/2 phosphorylation. The sham rats underwent aortic catheterization without occlusion. Parameters, including neurologic status, neuronal survival, inflammatory cell infiltration, and interleukin-1£] production in the spinal cords, were compared between groups. Results: The Tarlov scores in the (I/R) group were significantly lower than those in the (S), (P), and (P+I/R) groups on days 1, 3, 5, and 7. The numbers of surviving motor neurons in the (S), (P), and (P+I/R) groups were significantly higher than those in the (I/R) group. The (P) group exhibited higher spinal cord blood flow and tissue oxygenation after reperfusion than the (S) group. The (P+I/R) group exhibited higher spinal cord blood flow and tissue oxygenation within the first 60 min after reperfusion than the (I/R) groups. In the part 2 study, early ERK1/2 phosphorylation was observed after injury in the control group, followed by abundant microglial accumulation in the infarct area and increased interleukin-1£] expression. In the U0126 group, U0126 treatment completely blocked ERK1/2 phosphorylation. Microglial activation and spinal cord interleukin-1£] levels were significantly reduced. Neuronal survival and functional performance were improved. Conclusions: IPC ameliorates spinal cord I/R injury in rats, probably mediated by triggering spinal cord autoregulation and improving local spinal cord blood flow and tissue oxygenation. The ERK1/2 pathway may play a noxious role in spinal cord ischemia/reperfusion injury by participating in inflammatory reactions and cytokine production. According to our findings, these concepts may be the new therapeutic targets in patients requiring aortic surgery.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成24年2月29日 石黒久昌氏の博士論文として提出された

Abstract The first part of this research was focused on the relationship between injury severity and cell death mechanisms after spinal cord ischemia-reperfusion injury. The major blood supply to the thoracolumbar spinal cord comes from the segmental arteries originating from the thoracoabdominal aorta. Paraplegia cause by spinal cord ischemia is a devastating complication of thoracoabdominal aortic surgery. Previous studies indicated that ischemia-reperfusion injury of the central nervous system causes two distinct types of cell death, necrosis and apoptosis. It was also implicated that the intensity of injury can somehow affect the cell death mechanisms. In the first series of our experiments, by occluding the descending thoracic aorta with or without simultaneously inducing hypovolemic hypotension in rats, we established a model of experimental spinal cord ischemia-reperfusion (SCIR) in which the injury severity can be controlled. Recordings of carotid blood pressure (CBP) and spinal cord blood flow (SCBF) showed that aortic occlusion induced dramatic CBP elevation but SCBF drop in both the normotensive (NT) and hypotensive (HT) groups. However, the HT group demonstrated significantly lower SCBF during aortic occlusion, and much slower elevation of SCBF after reperfusion, and extremely poor neurological performance. Spinal cord lesions were characterized by infarction associated with extensive necrotic cell death, but little apoptosis and caspase-3 activity. In contrast, in the NT group, SCIR resulted in minor tissue destruction associated with persistently abundant apoptosis, augmented caspase-3 activity, and favorable functional outcome. The relative sparing of motoneurons in the ventral horns from apoptosis might have accounted for the minor functional impairment in the NT group. The severity of ischemia-reperfusion (I/R) injury was found to have substantial impact on the histopathological changes and cell death mechanisms, which correlated with neurological performance. These findings implicate that injury severity and duration after injury are two critical factors to be considered in therapeutic intervention. Based on the knowledge that bPrevious studies have implicated both excitotoxicity and apoptosis are involved in the pathogenesis of SCIR injury, we proposedtested the possibility that the N-methyl-D-aspartate (NMDA) receptor antagonist (dizocilpine maleate: (MK801) and the protein synthesis inhibitor (cycloheximide) would produce a synergic effect in the treatment of SCIR injury. In the second series of experiments, I/R iSpinal cord ischemia-reperfusion injury was induced by a thoracic aortic occlusion and blood volume reduction, followed by reperfusion and volume restoration. ischemia-reperfusion Rats were treated with vehicle, MK801, cycloheximide, or combination of MK801 and cycloheximide in combination. The MK801 and combined therapy group got a better recovery of hHind limb motor function recovery was better in the MK801 and combined-therapy groups than in the control and cycloheximide groups. On the 7th day after ischemia-reperfusion injury, all three treated groups showed significantly higher neuronal survival rates (NSR) than that of the control group. Among the three treated groups, the combined-treatment group showed the highest NSR. In addition, the Ttherapeutic effect of the combined-treatment group (27.4% increase of NSR) iwas better than the anticipated by the addition of MK801 and cycloheximide based on NSR data group. The number of apoptotic cells of was significantly reduced in the cycloheximide group and the combined-treatment group, as compared to that of the control group. It was unchanged in the MK-801 group. These results suggest that combined treatments directed at blocking both NMDA receptor-mediated excitotoxic necrosis and caspase-mediated apoptosis might have synergic therapeutic potential in reducing SCIR injury. Mitogen-activated protein kinases (MAPKs) including c-Jun N-terminal kinases (JNK), p38, and extracellular signal-regulated kinases (ERK), play important roles in the transduction of stressful signals and the integration of cellular responses. Although it has been generally held that the JNK and p38 pathways are related to cell death and degeneration, while the ERK pathway, cell proliferation and survival, controversy still exists. The roles of the ERK pathway in I/R injury of the CNS, in particular, remain to be clarified, because contradictory data have been reported by different investigators. Given this controversy, in the third series of experiments, we examined in injured spinal cords the temporal and spatial profiles of ERK1/2 activation following SCIR, and the effects of inhibiting the kinase that phosphorylates ERK1/2, MEK. The results showed that I/R injury induced an immediate phosphorylation of ERK1/2 in the spinal cord, which was alleviated by a MEK inhibitor, U0126. The control group was characterized by poorer neurological outcome, more severe tissue destruction, pronounced apoptosis, and lower neuronal survival. In contrast, the U0126-treated group demonstrated more apparent improvement of hind limb motor function, less tissue destruction, lack of apoptosis, and higher neuronal survival. In addition, administration of U0126 also significantly increased the activation of nuclear factor-£eB (NF-£eB) and the expression of cellular inhibitor of apoptosis protein 2 (c-IAP2). These findings implicate that the mechanisms underlying the neuroprotection afforded by ERK1/2 inhibition may be through the NF-£eB-c-IAP2 axis. The activation of the MEK-ERK signaling pathway appeared to be harmful in SCIR injury. Strategies aimed at blocking this pathway may bear potential therapeutic benefits in the treatment of SCIR injury. The second part of the research was focused on the pathophysiology of surgery-associated paraspinal muscle injury and measures to protect surgically violated paraspinal muscles. The wide dissection and forceful retraction of paraspinal muscles which are often required for posterior spinal sugery may severely jeopardize the muscles structurally and functionally. Immediate posteoperative pathological changes in the surgically violated paraspinal muscles may cause severe pain and a delay of patient ambulation. Long-term sequelae of surgical injury of paraspinal muscles include chronic back pain and impaired back muscle strength. Ironically, being a common complication of posterior spinal surgery, paraspinal muscle injury is so often neglected. Limited previous data indicate that the underlying pathophysiology of muscle damage involve both mechanical and ischemic mechanisms. We hypothesized that surgical dissection and retraction may produce oxidative stress within the paraspinal muscles. Meanwhile, we also proposed that the oxidative stress may trigger certain protective mechanisms within the insulted muscles. The first part of our study was a human study conducted to assess the significance of oxidative stress, and the relationship between it and the stress response mediated by heat shock protein 70 (HSP70) induction within paraspinal muscles under intraoperative retraction. A group of patients with lumbar spondylolisthesis treated with posterolateral lumbar spinal fusion, pedicle fixation and laminectomy were enrolled. Multifidus muscle specimens were harvested intraoperatively before, at designated time points during, and after surgical retraction. Muscle samples were analyzed for HSP70 and malondialdehyde (MDA) levels. Both HSP70 expression and MDA production within multifidus muscle cells were increased significantly by retraction. HSP70 expression then dropped after a peak at 1.5 hr of retraction, whereas MDA levels remained elevated even after release of retractors for reperfusion of the muscles. Histopathological and immunohistochemical evidence indicated that the decline of HSP70 synthesis within muscle cells after prolonged retraction was the result of severe muscle damage. These results highlighted the noxious impact of intraoperative retraction on human paraspinal muscles, and the significance of oxidative stress at the cellular and molecular levels. It is also implicated that intraoperative maneuvers aimed at reducing the oxidative stress within the paraspinal muscles may help attenuating surgery-associated paraspinal muscle damage. Given the findings of the first part of our study, and the knowledge that inflammation is a major postoperative pathological finding in surgically injured paraspinal muscles, we proceeded to examine the roles of two important inflammatory mediators, cyclooxygenase (COX)-2 and nuclear factor (NF)-£eB, in the pathogenesis of retraction-associated paraspinal muscle injury. A rat model of paraspinal muscle dissection and retraction that mimicks the conditions in human posterior spinal surgery was established. In the control group, paraspinal muscles were dissected from the spine through a dorsal incision, and then laterally retracted. Paraspinal muscle specimens were harvested before, and at designated time points during and after persistent retraction. The time course of NF-£eB activation as well as the expression of COX-2 were examined. Severity of inflammation was evaluated based on histopathology and myeloperoxidase (MPO) activity. NF-£eB activation was inhibited by the administration of pyrrolidine dithiolcarbamate (PDTC) in the PDTC-treated group. In the control group, retraction induced an early increase of NF-£eB/DNA binding activity in paraspinal muscle cells, which persited throughout the whole course of retraction. COX-2 expression was not detectable until 1 day after surgery, and reached a peak at 3 days. The time course of COX-2 expression correlated with that of inflammatory pathology and MPO activity. Extensive muscle fiber loss and collagen fiber replacement were observed at 7 days after surgery. Pretreatment with PDTC inhibited intraoperative NF-£eB activation and greatly downregulated postoperative COX-2 expression and inflammation in the muscles. Fibrosis following inflammation was also significantly abolished by PDTC administration. These findings indicate that NF-£eB-regulated COX-2 expression and inflammation play an important role in the pathogenesis of surgery-associated paraspinal muscle injury. Therapeutic strategies involving NF-£eB inhibition may be applicable to the prevention of such injury.

Thèse d'exercice : Médecine. Médecine générale : Paris 12 : 2005. / Titre provenant de l'écran-titre. Bibliogr. f. 79-81.

Thèse d'exercice : Médecine : Nancy 1 : 2008. / Titre provenant de l'écran-titre.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2003. / Vita. Includes bibliographical references (leaves 203-234).